Biogeochemical model of nitrogen cycling in Ahe (French Polynesia), a South Pacific coral atoll with pearl farming.

A biogeochemical model (ECO3M-Atoll) was configured to simulate the lower food web in Ahe Atoll lagoon where phytoplankton is mostly nitrogen limited. Understanding the dynamics of phytoplankton - the main food source for oysters - is crucial for the management and the allocation of new pearl farming sites. After parametrizing the model with in situ observations, we tested different hypotheses about nitrogen cycling (benthic remineralization, atmospheric N fixation, etc.) and compared the results to a large observational dataset. Model results show that simulated (pico- and nano-) phytoplankton biomass and nitrogen concentrations are close to in situ data. The simulated biogeochemical processes (uptake and primary production) are also very similar to the observed values. In the model, primary production ranged from 1.00 to 2.00 mg C m-3 h-1 for pico- and 0.40 to 1.00 mg C m-3 h-1 for nanophytoplankton; mean N uptake was 2.02 µmol N m-3 h-1 for pico- and 1.25 µmol N m-3 h-1 for nanophytoplankton.

Validation of a new metabolic marker to assess the vascular viability of vitrified whole sheep ovaries.

BACKGROUND: Whole ovary cryopreservation has been suggested as a means to preserve fertility. In animal models, autologous cryopreserved ovary transplants frequently undergo thrombosis and a method to assess the vascular viability of cryopreserved ovaries would be valuable. We developed a staining method using methylthiazolyl blue tetrazolium (MTT, a metabolic marker) to assess the pedicle metabolism of whole ovaries vitrified using cryoprotectant called 'VS4'. METHODS: Whole sheep ovaries were perfused with MTT (1 g/l). In one group, ovarian tissue lesions were induced by immersing the ovarian pedicle in medium at 53°C or 65°C or in liquid nitrogen prior to MTT perfusion. In the second group, several metabolic substrates (d-glucose, l-glucose and pyruvic acid) and inhibitors [2-deoxy-d-glucose for d-glucose metabolism, azide for mitochondrial respiration and diphenyleneiodonium (DPI) for NADPH oxidase (an effector of the pentose phosphate pathway)] were added to the MTT stain. The third group was subjected to VS4 ± vitrification/warming prior to MTT perfusion. Pedicle MTT staining was assessed qualitatively by histological examination of frozen sections or quantified at 564 nm after solubilization in alcohol. RESULTS: MTT strongly and reproducibly stained the vascular smooth muscle. Heating at 53°C or 65°C or cooling in liquid nitrogen significantly diminished MTT staining by 48% (P = 0.001, n = 10), 94% (P = 0.0002, n = 10) and 94% (P = 0.0002, n = 10), respectively. MTT staining was affected by d-glucose metabolism: absence of d-glucose, substitution of unmetabolized l-glucose for d-glucose or addition of 2-deoxy-d-glucose significantly decreased MTT staining by 44% (P < 0.01, n = 10), 45% (P < 0.01, n = 10) and 29% (P < 0.01, n = 10), respectively. Pyruvic acid failed to correct the MTT staining decrease induced by d-glucose deprivation and azide did not decrease MTT staining, suggesting that MTT staining could be independent of mitochondrial metabolism. Adding DPI significantly inhibited MTT staining by 25% (P < 0.001, n = 10), suggesting involvement of the pentose phosphate pathway's effectors. Compared with controls, VS4-vitrified/warmed pedicles showed significantly less MTT staining (-30%, P < 0.005, n = 10), with unstained foci, whereas unvitrified VS4-exposed pedicles showed no difference. CONCLUSIONS: MTT can serve as a qualitative and quantitative vascular viability marker.VS4 vitrification caused alterations in ovarian vascular metabolism. MTT staining should allow accurate comparisons of whole-organ cryoprotection protocols.

Technical aspects of laparoscopic ovarian autograft in ewes after cryopreservation by slow-cooling protocol.

Iatrogenic ovarian failure and infertility are long term-term side effects of anticancerous gonadotoxic treatments in children or women of reproductive year. Ovarian cortex cryopreservation can be a solution to preserve immature germinal cells before gonadotoxic treatment, for later transplantation. The aim of our study was to prove the efficiency of a laparoscopic technique for orthotopic graft after a slow-freezing/thawing protocol, and to evaluate the effect of ovarian cryopreservation and autograft on the primordial follicle survival rate. Experimental surgical study was performed on 6- to 12-month-old ewes. The study was approved by the ethic committee of the Lyon-veterinary-school. The left ovary was removed by laparoscopy and cut in half, and medulla was excised. In group 1 (n = 6), autograft was performed immediately on the right ovary, and in group 2 (n = 6), graft was performed after a slow-freezing/thawing protocol. The second hemi-ovary served as an ungrafted control fragment. A polypropylene/polyglactin mesh was included between graft and base to separate the two structures, to help histological analysis. The mean graft performance time was 71 +/- 8 min in the first group and 57 +/- 10 min in the second. Freezing did not affect the number of primordial follicles. In the ungraft control fragments, the global anomaly rate (cytoplasm plus nuclear anomaly) increased after freezing (p < 0.05). Others results did not reach signification. Pelvic adhesion occurred only once. The post-graft primordial follicle survival rate was 5.1 +/- 2.8% in the non-frozen group vs. 6.3 +/- 2.3% after freezing/thawing. Kruskal-Wallis and Wilkoxon non-parametric tests were used for statistical analysis. Laparoscopy seems to be a well-adapted technique for ovarian tissue orthotopic autograft. The main follicle loss occurs before graft revascularization. Our orthotopic graft's procedure has to be improved to obtain a better graft's neovascularization, and to have a better long-term post-graft primordial follicle survival rate.

[Vitrification: a future technique for ovarian cryopreservation? Physical basis of cryobiology, advantages and limits]. / La vitrification : technique d'avenir pour la cryoconservation ovarienne ? Bases physiques de cryobiologie, avantages et limites.

Ovarian cryopreservation is presently indicated in patients who undergo a gonadotoxic treatment, most commonly for anticancer procedures. These procedures can strongly alter fertility by damaging the follicular ovarian reserve. Although six human live births have been described in the world after ovarian tissue cryopreservation and autografting, the techniques of cryopreservation techniques are not consensual. Vitrification is a physical process that allows cryopreservation without formation of ice crystals, by transformation of a highly concentrated solution in a glassy or amorphous state. Vitrification is at present rapidly expanding in the biology of reproduction. With the classic methods of freezing, formation of ice crystals within the ovarian tissue is systematic and can entail cellular lesions. Which is why more and more teams question the theoretical advantage of the vitrification for ovarian cryopreservation. Our objective was to summarize the fundamental physical basis of cryobiology, necessary for an understanding of vitrification. From our experience, we also wanted to point out the practical difficulties of this technique, and we are proposing a model of evaluation and validation that uses differential scanning calorimetry, applicable to any protocol of vitrification.

[State of the art on in vitro folliculogenesis in mouse]. / Etat actuel de la folliculogenèse in vitro chez la souris.

Follicle culture systems have been developed so as to achieve in vitro fertilization of oocytes coming from immature follicles. The in vitro folliculogenesis methods would be especially useful in reproductive medicine to restore fertility in women having undergone ovarian cryopreservation. Several culture systems allowing in vitro growth of small follicles have been developed in mouse. These have proven to be successful by the birth of healthy offsprings. Some elements determine the outcome of culture: follicle isolations at a defined stage of development, follicular morphology preservation, and supplementation of growth factors or hormones. Development of follicle culture in the mouse model led to a better understanding of ovarian physiology, in particular the relation between endocrine and paracrine factors on follicle development. The in vitro techniques in mouse became a valuable tool for improving reproductive technics improvement, and for toxicology studies.

Towards whole sheep ovary cryopreservation.

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 degrees C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12-16 degrees C/min from -196 to -133 degrees C) in liquid nitrogen vapour, followed by rapid thawing in a 45 degrees C water bath at about 200 degrees C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4+/-2.2% of small follicles were viable while histological analysis showed that 48+/-3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.

[Ovarian tissue vitrification: Cortex and whole ovary in sheep]. / Vitrification du tissu ovarien: cortex et ovaire entier chez la brebis.

OBJECTIVE: The aim of this study was to evaluate a cryopreservation technique by vitrification of cortex or whole ovaries in sheep, using two cryoprotectant solutions: VS1 and VS4 and to study their physical properties to avoid ice crystallisation by vitrification of whole sheep ovaries permeated with a cryoprotectant solution. ANIMALS AND METHODS: From 6-month-old ewes, whole sheep ovaries with their vascular pedicles were collected at the slaughterhouse or at the veterinary school and prepared for cryoprotectant toxicity tests and freezing procedure. Follicle viability was measured by trypan blue test and histological examination of ovary. The hemi-ovarian cortex was stored in liquid nitrogen. Four to six weeks after the first laparotomy, the controlateral ovary was removed and the vitrified-warmed hemi-ovary was sutured. Thermal properties of a cryoprotectant solution called VS4 (critical cooling rates [Vccr], vitreous transition temperature [Tg], end of melting temperature [Tm]) were measured by differential scanning calorimetry. RESULTS: No significant difference in follicle viability or normal follicle rates was observed between ovarian cortex exposed or non-exposed to cryoprotectant solutions. Nor was any significant difference observed before and after vitrification. Three pregnancies occurred, from which four lambs were born after autografts of vitrified ovarian cortex. With whole ovary, the decrease in the number of normal follicles was lower when frozen-thawed ovaries were treated with VS4 (P = 0.04). There were less nuclear anomalies (P = 0.02). The Vccr of VS4 has been estimated to be 14.3+/-1.1 degrees C/min and Tg was -125.0+/-0.2 degrees C. Because the penetration of cryoprotectants was very low, Vccr was very high and the cooling speed did not allow cortex to vitrify. DISCUSSION AND CONCLUSIONS: Cryopreservation of cortex or whole ovary by vitrification seems a promising technique in reproductive medicine. The best histologic results were obtained with the VS4 cryoprotectant when whole ovary was vitrified.